Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.7.7 (DNA polymerase)
17,007 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Hepatitis B core antigen (HBcAg) particles, approximately 27-28 nm in diameter and rho = 1.30-1.35 g/cm3, were purified from the liver of a chimpanzee experimentally infected with hepatitis B virus (HBV) while under cyclophosphamide treatment. The purified HBcAg particles incorporated radioactive deoxythymidine triphosphate. The product was precipitable by trichloroacetic acid and sensitive to DNase, but resistant to digestion by RNase. The reaction required four deoxyribonucleosise triphosphates- dATP, dCTP, dGTP and dTTP. Exogenous template did not enhance the reaction. From these findings, it was suggested that HBcAg particles purified from the HBV-infected chimpanzee liver contained DNA polymerase and endogenous DNA.
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PMID:Hepatitis B core particles with endogenous DNA polymerase activity from chimpanzee liver. 68 Nov 46

The Bacillus subtilis dnaF (polC) gene that codes for the alpha subunit of the DNA polymerase III holoenzyme has been sequenced. It consists of 4005 base pairs coding for 1335 amino acids (from the start to the stop codon), giving a molecular weight of 151,273. A mutation (azp-12) that confers resistance to the antimicrobial drug 6-(p-hydroxyphenylazo)-uracil is due to a single base change at nucleotide 3523, from TCA to GCA, resulting in a change of the 1175th amino acid, serine, to alanine. It is in the active site and located at the C-terminal part of the enzyme. The amino acid composition in an N-terminal domain has 26% homology to the epsilon subunit coded by the dnaQ gene of Escherichia coli, which is a 3'----5' proofreading exonuclease, supporting an earlier observation that this function is an integral part of the polymerase molecule in B. subtilis.
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PMID:DNA polymerase III gene of Bacillus subtilis. 249 83

The 31mer 5'-TCA ACG CTA GAA TTC GGA TCC ATC GCT TGG T, the complementary 33mer 5'-CCA AGC GAT GGA TCC GAA TTC TAG CGT TGA GAT, the 40mer 5'-GGC CAG GAT GGT GAA GAA TTC GAT CCG GTA CGT AGC TAA G, and the complementary 42mer 5'-TAC TTA GCT ACG TAC CGG ATC GAA TTC TTC ACC ATC CTG GCC were synthesized and their reactivity towards EcoRI was studied. It was found that the 31mer and the 40mer were cleaved at a comparable rate to the 31mer-33mer hybrid and the 40mer-42mer hybrid, respectively. The rate of cleavage of the 33mer and the 42mer was an order of magnitude lower. To rule out possible intermolecular duplex formation, the 33mer was immobilized on cellulose by ligation and labeled with alpha 32P-dCTP using Klenow fragment of E. coli DNA polymerase. EcoRI cleaved this immobilized oligomer into specific fragments.
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PMID:Cleavage of single stranded oligonucleotides by EcoRI restriction endonuclease. 302 89

An approach to the investigation how growth factors and hormones regulate mammalian cell proliferation is to study the activity of enzymes involved in DNA replication. Quiescent cultures of Swiss mouse 3T3 cells were stimulated with prostaglandin F2 alpha, insulin, and/or hydrocortisone for a time at which less than 50% of the cells had initiated DNA synthesis. Such cells were lysed with a Ca++-containing hypotonic buffer and incubated with a nucleotide mixture including [3H]thymidine-triphosphate for 1 hr at 37 degrees C. The amount of radioactive label incorporated into the trichloroacetic acid (TCA)-precipitate and the percentage of labeled nuclei correlated with the in vivo stimulation. Analysis of radioactively and density-labeled DNA in sucrose and CsC gradients indicated that the incorporation of label reflected semiconservative replication. DNA polymerase activities were assayed in supernatants from whole-cell lysates prepared with a hypotonic buffer not containing Ca++. Using various templates, it was shown that the increase in activity of DNA polymerase alpha correlated with the percentage of cells in S phase upon the different stimulation, while DNA polymerase beta activity after various times of stimulation showed that this activity increased only when cells began to enter S phase, regardless of the combination of growth factor and hormones.
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PMID:Stimulation of DNA replication by growth factor and hormones in Swiss 3T3 cells: comparison of the rate of entry into S phase with in vitro DNA synthesis and DNA polymerase alpha activity. 327 79

Oil-induced guinea pig peritoneal exudate macrophages were found to incorporate 3H-thymidine into trichloroacetic acid-insoluble fraction. In pulse-labeling experiments, the incorporated 3H-thymidine was detected in short fragments of DNA, which corresponded to the Okazaki fragments. These results indicate that the observed thymidine incorporation is due to nuclear DNA replication but not DNA repair. The observed DNA synthesis of the macrophages was remarkably suppressed when the cells were cultured in a presence of muramyl dipeptide (MDP) or bacterial lipopolysaccharide (LPS). The significant decrease of DNA polymerase alpha activity was found in the cells treated with MDP or LPS. In contrast, the activity of polymerase beta was not at all affected by the same treatment.
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PMID:Preferential loss of DNA polymerase alpha following suppression of replicative DNA synthesis of guinea pig macrophages by the immunostimulants muramyl dipeptide or lipopolysaccharide. 346 95

Guinea pig peritoneal exudate macrophages actively incorporated [3H]thymidine into trichloroacetic acid-insoluble fraction in vitro. The incorporation of [3H]thymidine was almost completely inhibited by aphidicolin, an inhibitor of DNA polymerase alpha and an autoradiograph showed heavy labeling in nuclei of 15% of macrophage populations. These results indicate that the observed thymidine incorporation was due to a nuclear DNA synthesis. The [3H]thymidine incorporation was markedly suppressed when macrophages were activated by immunoadjuvants such as muramyl dipeptide (MDP) or bacterial lipopolysaccharide (LPS). The suppression of [3H]thymidine incorporation by MDP was neither due to the decrease in thymidine transport through the cell membrane, nor due to dilution by newly synthesized "cold" thymidine. An autoradiograph revealed that MDP markedly decreased the number of macrophages the nuclei of which were labeled by [3H]thymidine. These results suggest that the suppression of [3H]thymidine incorporation by the immunoadjuvants reflects a true inhibition of DNA synthesis. The inhibition of DNA synthesis by MDP was also observed in vivo. Further, it was strongly suggested that the inhibition was not caused by some mediators, such as prostaglandin E2, released from macrophages stimulated by the immunoadjuvants but caused by a direct triggering of the adjuvants at least at the early stage of activation. Cyclic AMP appears to be involved in the inhibitory reaction.
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PMID:Inhibition of macrophage DNA synthesis by immunomodulators. II. Characterization of the suppression by muramyl dipeptide or lipopolysaccharide [3H]thymidine incorporation into macrophages. 643 15

A modified and improved technique for the detection of hepatitis B virus-specific DNA polymerase activity is described. DNA polymerase is released from Dane particles by mixing samples with the detergent Nonidet P-40 and beta-mercaptoethanol. After incubation of pretreated samples with a reaction mixture containing tritiated thymidine-methyl-5'-triphosphate (3H-TTP), DNA is precipitated onto a trichloroacetic acid (TCA)-treated paper. Unincorporated 3H-TTP is then chromatographically eluted with a 5% TCA solution and precipitated counts are determined. A sample is considered positive for DNA polymerase if the incorporated counts are significantly higher than the counts of a group of negative control samples. The modifications include pretreatment of the paper with TCA, chromatographic elution of unincorporated 3H-TTP with TCA solution, prefiltration of the sample through bacteriological filters, and use of sound statistical methods for evaluation of data. These changes have led to a highly reproducible, reliable and sensitive technique. The coefficient of variation of negative control samples from various test runs was in the range of 2.7-8.5%. A linear relationship between incorporated counts and DNA polymerase concentration was shown. A total of 419 serum samples from asymptomatic HBsAg-carrying blood donors were tested. Twenty-three (5.5%) of these were found to contain detectable DNA polymerase activity. All 23 samples also contained HBeAg.
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PMID:A modified technique for the detection of hepatitis B virus-specific DNA polymerase. 702 73

We demonstrate here that stilbene estrogen (diethylstilbestrol) is converted to nuclear protein binding metabolite(s) both in vitro and in vivo. In vitro reaction of DES with nuclei from hamster liver or kidney in the presence of cumene hydroperoxide or NADPH revealed binding of [3H]DES in nuclear proteins (histones; nonhistones precipitable by 2% TCA, NH2; nonhistones soluble in 2% TCA, NH30). The binding was significantly inhibited by cytochromes P450 inhibitors. In an in vitro system [3H]DES quinone, one of the metabolites of DES, was able to bind to pure nonhistone proteins RNA polymerase and DNA polymerase. The binding of [3H]DES quinone to nonhistones RNA polymerase and DNA polymerase was inhibited by low molecular weight thiols, i.e. glutathione and cysteine, or thiol modifiers, such as n-ethylmaleimide, dithionitrobenzoic acid and hydroxymercuric benzoate. DES and DES metabolites inhibited transcriptional activity. In vivo [3H]DES was able to bind to nuclear proteins of hamster liver, kidneys and testes. The level of in vivo [3H]DES binding to all three types of nuclear proteins (histones, NH2, NH30) in the kidney (target organ) was two or more fold higher than that observed in the liver or testis (nontarget organs). Four nuclear NH30 proteins (mol wts.: 56, 37, 33 and 28 kDa) were irreversibly bound to [3H]DES in vivo. The in vivo binding of [3H]DES to transcriptionally active chromatin NH30 proteins also was observed. The data reported here establish that DES was able to bind to liver or kidney nuclear proteins in vitro, which was catalyzed by nuclear enzymes when fortified with an appropriate cofactor. DES quinone may be one of the protein binding metabolites. DES and DES metabolites inhibited transcriptional activity. The level of in vivo binding of [3H] DES to nuclear proteins of kidney (target organ) was double in comparison with that observed in liver or testis (nontarget organs). In vivo modifications in the chromatin proteins may be a factor in the development of DES-induced renal carcinogenesis is not clear.
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PMID:In vivo binding of diethylstilbestrol to nuclear proteins of kidneys of Syrian hamsters. 773 58

A single polypeptide of approx. 67 kDa mol.wt. with DNA polymerase activity has been chromatographically purified from shoot tips of 72 hr. grown germinated seedlings of rice (Oryza sativa.L.cv IR-8). An approx. 4800 fold enrichment of specific activity was measured by the incorporation of 3H-dTMP into trichloroacetic acid insoluble fraction, using activated calf thymus DNA as template-primer. The enzyme uses different types of DNA but not RNA as a template. The enzyme requires Mg+2, high KCl, and is highly sensitive to dideoxyribonucleoside triphosphate but is unaffected by aphidicolin, suggesting a plant counterpart of mammalian DNA polymerase beta. Replication of M13mp18 single stranded DNA by extension of 5'32P-labeled 17-mer primer(-40) showed distributive type of DNA synthesis by the rice DNA polymerase beta, which is a characteristic feature of mammalian DNA polymerase beta.
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PMID:Isolation of mammalian pol beta type DNA polymerase in shoot tips of germinated seedlings of IR-8 rice (Oryza sativa L.). 879 34

Assuming that the efficiency of the incorporation of 5-methyl-2'-deoxyisocytosine-5' triphosphate (dMiCTP) and dTTP opposite isoguanine (iG) is a measure of the proportion of the keto and enol tautomers of iG in the Thermus aquaticus DNA polymerase active centre, we studied the influence of temperature and iG-neighbouring bases in the template on base-pairing of iG. On the basis of experiments with four sequences (3'-TXT-5', 3'-GXG-5', 3'-CXC-5', 3'-CXT-5', where X=iG) at 37, 50, 65 and 80 degrees C, we found that 3'-neighbours decrease the fraction of the keto tautomer in the order C>G>or=T, whereas temperature apparently does not influence the tautomeric equilibrium of iG. The variability of the ratio of incorporation of dMiCTP versus dTTP (5-20) primarily reflects the variability of K (m) values, since V (max) values are roughly similar, which indicates that the iG.MiC and iG.T pairs fit the polymerase active centre equally well. The altering of the base-pairing of iG by sequence context is discussed in relation to tautomerism and miscoding of this oxidized adenine derivative. A key derivative for preparation oligodeoxynucleotides, O (2)-diphenylcarbamoyl- N (6)-[(dimethylamino)ethylidene]-2'-deoxyisoguanosine, is extremely labile (t (1/2)=3.5 min) in 3% trichloroacetic acid/dichloromethane, i.e. under the conditions of automated DNA synthesis, which results in low yield and length heterogeneity of templates.
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PMID:Neighbouring bases in template influence base-pairing of isoguanine. 1238 28


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